More than a million people per year die by suicide worldwide, more than all homicides and deaths in all wars, making suicide a global health problem. Many people with Major Depressive Disorder (MDD), a serious disorder with an enormous economic burden, die by suicide. In suicides, we find more tryptophan hydroxylase (TPH2), the serotonin (5-HT) biosynthetic enzyme, as well as more TPH+ neurons and hypothesize this is compensation for deficient 5-HT neurotransmission. We now have a group of unmedicated MDD nonsuicides to allow us to determine whether the changes we find are associated with the diathesis of suicide or with MDD. We propose a study of matched triplets (n=15) of an MDD suicide, an MDD nonsuicide and a nonpsychiatric nonsuicide control, all unmedicated and characterized psychiatrically. We aim to elucidate molecular mechanisms involved in regulating TPH2 expression and function in the dorsal raphe nucleus (DRN) and in the prefrontal cortex (PFC) and separate the effects of MDD from suicide. We hypothesize that the paradox of low 5-HT neurotransmission in the PFC and high TPH2 in the DRN in suicides is a consequence of dysregulation of cAMP/PKA dependent and GSK3beta pathways regulating TPH2 phosphorylation in the DRN and signaling in the PFC. In Specific Aim 1, we will use in situ hybridization and immunoautoradiography to measure the amount of TPH2 mRNA and protein in the DRN. We hypothesize that MDD suicides will over-express TPH2 compared to the nonsuicide MDD and control groups. Alternatively, TPH2 expression in suicides may have anatomically restricted changes compared to MDD non-suicides, similar to our previous findings of widespread 5-HT transporter reductions in MDD but restricted changes in suicide. In Specific Aim 2, Western blots will be used to determine the levels of TPH2 and phosphorylated-TPH2 (p-TPH2) as well as key kinases involved in TPH2 regulation in both the brainstem and the dorsolateral and ventral PFC. We hypothesize that PKA expression will be lower in suicides, resulting in less phosphorylation of TPH2 and that suicides will have higher GSK3beta expression associated with the lower TPH2 function and higher AKT and p-AKT to inactivate GSK3beta. This defect will be widespread in the DRN in MDD, but greater in magnitude and with a different anatomy in MDD suicides. Alternatively, PKA expression may be elevated in suicides in the DRN and PFC in association with autoreceptor upregulatory changes. In Specific Aim 3, HPLC will be used to measure 5-HT, tryptophan and 5-HIAA in the brainstem and the PFC. We hypothesize that suicides will have more 5-HT and 5-HIAA compared to controls or MDD nonsuicides. Alternatively, 5-HT and 5-HIAA may be less in MDD. This will suggest that the serotonergic deficiency associated with suicide is different from MDD and lies, not in 5- HT synthesis, but in downstream processes such as reduced transmitter release in the PFC. Thus, the serotonergic deficiency profile in suicide will be distinct from that in MDD.